Epidemiological evidence points to the value of cardiac glycosides like digoxin and digitoxin for treatment of breast cancer and recent reports suggest they may be especially useful to treat non-small cell lung cancer. These and other findings underlie our belief that a cancer drug could be developed from such natural products. A major need in the development of a cancer drug lead will be to maximize the difference between inotropic activity, which could be cardiotoxic, and antitumor activity due to other effects of such glycosides. We will aim to discover a new drug for cancer treatment by optimizing the novel cardiac neoglycoside leads found in a cancer drug discovery program at the University of Wisconsin. The necessary IP to pursue this goal has been exclusively licensed to Centrose by the university. Two leads derived from digitoxigenin have already been found with nanomolar potency and high selectivity in cancer cell cytotoxicity tests, revealing an unexpected but potentially very useful influence of sugar structure on biological activity. They exhibit a >18-fold safer therapeutic index than digitoxin, based on their much weaker inhibition of the human Na/K-ATPase in a cell based assay. Synthesis of additional cardiac neoglycosides and comparison of their cytotoxicity towards cancer cell lines and inhibition of the Na/K-ATPase transporter in the cell based assay will therefore be the primary assays used to assess the potency and therapeutic index. The synthetic studies will involve surveying new cardenolide aglycones that carry a subset of the original library of hexoses and pentoses; and carrying out post-neoglycosylation chemistry at selected positions in the attached sugars using old and new leads. Secondary assays will involve (i) cell-based tests of potential cardiotoxicity due to long QT prolongations resulting from inhibition of hERG channel function or trafficking and to inhibition of repolarization of cardiomyocytes made from human embryonic stem cells, (ii) determination of effects on intracellular calcium ion transients in animal cardiomyocytes, because this is known to be correlated with cardiac arrhythmias in humans, and (iii) determinations of the effect of new analogs on induction of cancer cell apoptosis mediated through the Src/EGFR/ERK and TNF-alpha receptor/NFkB signalling pathways. Our goal will be to identify analogs with a better apparent therapeutic index than the current leads. The antitumor activity of two optimized leads chosen on the basis of the results of these studies will be determined by a contract research organization in xenograft mouse models of relevant human tumors. The research will aim to discover a new type of drug for cancer treatment that is based on chemical variations of the sugar components of cardiac glycosides that have been used for centuries to treat congestive heart failure. In the present study the goal will be to identify drug leads that have the greatest safety index between antitumor activity and potential cardiotoxicity. [unreadable] [unreadable] [unreadable]